Contamination of the environment with metal ions and/or alkyl and thiol derivatives of metals has increased over the last several decades, with toxic levels of the contaminants being reached in air, water and/or soil in certain locations. Contamination may stem from human and industrial sources, or in certain locales, the soil is contaminated naturally with such toxic metals as arsenic, cadmium, copper, cobalt, lead, mercury, selenium and/or zinc.
Mercury is often found in soil and marine sediments as thiol salts, as chelates with acidic humic substances such as methylmercury and to a lesser extent other organomercurials, and as free Hg.sup.++. Mercury cycles through the aqueous phase and into the atmosphere as volatile elemental Hg and methylmercury, and is then oxidized and washed by rain into the marine environment [Barkay et al. (1992) Biodegradation 3:147-159]. Some bacteria in soil and sediments can detoxify ionic mercury by reducing it to its metallic form in an NADPH-coupled reaction, which is efficiently catalyzed by mercuric ion reductase. Mercury is often found bound in the form of organomercurial compounds in contaminated animals and microbes [Barkay et al. (1992) supra; Robinson and Tuovinen (1984) Microbiological Reviews 48:95-124]. In fish, where mercury toxicity is well studied, most of the tissue-associated mercury is found as methylmercury, and its production may be the product of a nonenzymatic reaction of Hg.sup.++ with methyl-B12 [Pan Hou and Imura (1987) Arch. Microbiol. 131:176-177]. Dimethylmercury is volatile, and both mono- and dimethylmercury are extremely toxic [D'Itri and D'Itri (1987) Environ. Management 2:3-16]. Although the effects and levels of methylmercury in plants and the contribution of plants to the production of organomercurial compounds in the environment are not known, it is likely that macrophytes are a major source of organomercury compounds in the environment based on the biochemical activity of the dominant macrophytes in many fresh water, estuarine and marine environments.
Certain plants express phytochelatins, a group of .gamma.-glutamylcysteine peptides which are the products of a complex synthetic pathway [Scheller et al. (1987) Plant Physiol. 85:1031-1035]. Phytochelatins mediate some metal resistance in plants which produce them [Grill et al. (1987) Proc. Natl. Acad. Sci. USA 84:439-443]. Lefebvre et al. (1987) Biotechnology 5:1053-1056 reported the construction of transgenic plant tissue expressing a mammalian metallothionein gene; that tissue exhibited some resistance to cadmium.
The bacterial gene merA used by the present inventors is derived from the transposon Tn21, which was originally isolated from the Incompatibility Group IncFII resistance plasmid NR1 [see e.g., Gilbert and Summers (1988) Plasmid 20:127-136]. The product of the bacterial merA gene is mercuric ion reductase (MerA). MerA can detoxify ionic mercury by reducing it to its less toxic (insoluble and volatile) elemental form (Hg.sup.0). MerA belongs to a family of reductase enzymes which are related in their primary structures. As a family, these reductases act on a wide variety of organic and thiol substrates in addition to the thiol salts of divalent Hg.
Some of the bacterial mer operons also encode an organomercurial lyase (MerB, methylmercury lyase, Tn21 merB gene product) which catalyzes the protonolytic cleavage of carbon-mercury bonds, RCH.sub.2 Hg.sup.+ .fwdarw.Hg.sup.++ +RCH.sub.3, and together with MerA produces what is termed broad spectrum mercury resistance (resistance to both thiolmercurial and alkylmercurial compounds and resistance to mercuric ion). The MerB protein cleaves a variety of carbon-mercury compounds, from methylmercury to long chain hydrocarbon and aromatic derivatives [Begley et al. (1986) Biochemistry 25:7186-7192; Begley et al. (1986) ibid. 7192-7200]. This process removes methylmercury and then metallic mercury from the environment.
Additional genes often part of bacterial mer operons include merT (mercury transport through the cell membrane) and merP (mercury sequestration in the periplasmic space of gram-negative bacteria). Mercury resistance genes are reviewed in Summers, A. O. (1986) Ann. Rev. Microbiology 40:607-634.
Regions which are naturally contaminated with heavy metals are often characterized by scrubby heavy-metal tolerant vegetation [Brooks and Malaisse (1985) The Heavy Metal-tolerant Flora of South Central Africa, A. A. Balkema Press, Boston, Mass.; Wild, H. (1978) "The Vegetation of Heavy Metal and Other Toxic Soils," in Biogeography and Ecology of Southern Africa, Wergren, M. J. H., ed, Junk, The Hague, Netherlands]. Certain of these naturally occurring metal-resistant plants hyperaccumulate large amounts of heavy metals in the form of malate or citrate chelates. These plants have been found in a variety of habitats, but often they exhibit bizarre metal ion requirements, grow poorly in less exotic habitats, and are of little direct economic value as crop or forest species.
There is a long felt need in the art for the in situ remediation of toxic metal ions and/or metal complexes (e.g., alkyl and thiol metal adducts). The present invention enables phytoremediation and/or revegetation of contaminated environments via the plant-expressible metal resistance coding sequences disclosed herein.